Proteins of the Tumor Necrosis Factor (TNF) superfamily, such as Fas ligand (FasL) induce programmed cell death (apoptosis) upon ligation with relevant receptors on the cell surface (Fas). Because the anticancer action of chemotherapeutic agents is in large part attributable to their ability to induce apoptosis and Fas-resistant tumors show cross resistance to drug-induced apoptosis in vitro, we made the hypothesis that tumors with a defective Fas/FasL signaling pathway are refractory to treatment and exhibit a more aggressive behavior. We have investigated the various components of the Fas/FasL pathway in the Ewing's sarcoma family of tumors (ESFT) and in neuroblastoma (NB), in order to understand the molecular events that lead to their impaired apoptosis and hence defective response to chemotherapeutic agents leading to treatment failures. This approach is expected to lead to the discovery of novel therapeutic agents in ESFT and NB. We had previously shown that Ewing?s sarcoma cells express Fas and FasL (Mitsiades N. et al, Am. J. Pathol. 153:1947-1956, 1998), but are not always sensitive to Fas-mediated apoptosis. Recently (Mitsiades N. et al, Cancer Res. 61:2704-2712, 2001), we found that the newly described TNF-Related Apoptosis Inducing Ligand (TRAIL) kills even Fas-resistant Ewing?s sarcoma cells in vitro through a caspase-mediated pathway which depends on the surface expression of at least one of the two TRAIL death receptors (DR4 and DR5). We also found that Ewing?s sarcoma tissues frequently express the DR4 and/or DR5 receptors, and therefore, Ewing?s sarcoma cells are likely to respond to TRAIL in vivo. This novel finding opens up the possibility of using TRAIL in the treatment of recurrent or metastatic Ewing?s sarcoma patients, for whom current therapeutic modalities have failed. We had previously shown that in Ewing?s sarcoma cell lines, Fas- and drug-induced apoptosis is enhanced by treatment with synthetic matrix metalloproteinase (MMP) inhibitors which inhibit the cleavage of FasL into the less potent soluble form, thus inducing accumulation of the potent full length molecule on the cell surface (Mitsiades N., et al, J. Natl. Cancer Inst. 91:1678-1684, 1999). Recently in collaboration with Dr. Stamenkovic at Harvard Medical School (Mitsiades N., et al, Cancer Res. 61:577-581, 2001), we showed that MMP-7 which is broadly expressed in Ewing?s sarcoma cells and tissues is responsible for the cleavage of FasL in Ewing?s cells. Exogenous and tumor cell-derived MMP-7 inhibited Fas- and drug-induced apoptosis in these cells. These data make MMP-7 a potential therapeutic target, because its inactivation may enhance the efficacy of conventional chemotherapy in Ewing?s sarcoma. In a recent study (Poulaki V., et al, Cancer Res. 61:4864-4872, 2001) we have identified the defects in the Fas pathway that lead to apoptosis resistance in neuroblastoma cells. Namely, we found that in neuroblastoma, Fas-mediated apoptosis is mitochondria-dependent and is inhibited by the FLICE (caspase 8) inhibitory protein (FLIP) and bcl-2. We have also identified, a novel mechanism of action for bcl-2 through binding and sequestration of caspase 8 and thus inhibition of its activation upstream of mitochondria. Having identified the inhibitory proteins and the mechanisms by which they inhibit apoptosis, we introduced the possibility of gene-targeting therapies for bcl-2 and FLIP which may prove useful in the treatment of drug-resistant neuroblastomas when used in combination with traditional chemotherapeutic regimens. MYCN amplification is one of the single most powerful prognostic factors in neuroblastoma. Recent studies have confirmed that most children with MYCN-amplified neuroblastoma have rapid disease progression and die regardless of treatment. Yet, other studies have shown that MYCN sensitizes human neuroblastoma cells to interferon- or drug-mediated apoptosis. We have shown (Poulaki V., et al, Proc. Am. Assoc. Cancer Res. 41: 416, abstract, 2000) that MYCN upregulates the transcriptional activity of the FasL promoter in neuroblastoma cells through direct binding with two canonical enhancer boxes in the FasL promoter. We also found higher FasL expression in MYCN-amplified neuroblastoma cells in vivo. FasL is used by Fas-resistant tumor cells to kill activated immune cells thus leading to tumor aggressiveness. In keeping with this concept, we found that neuroblastoma cells with MYCN amplification which also had higher levels of FasL induced lymphocyte cell death at a higher level than neuroblastoma cells without MYCN amplification. The phenomenon was inhibited by FasL blocking antibodies.These data (manuscript in submission) support FasL upregulation as one of the mechanisms by which MYCN confers aggressiveness to neuroblastoma cells. They also explain why MYCN amplification sensitizes some neuroblastoma cells (those with a functional Fas pathway) to Fas- and drug-induced apoptosis, and in combination with our data above, suggest that MYCN-amplified neuroblastoma cells may be more vulnerable to chemotherapeutic agents than single copy MYCN neuroblastoma cells, once their downstream Fas pathway becomes functional through downregulation of bcl-2 and FLIP. Our current preliminary data have proven this hypothesis correct, because MYCN-overexpressing neuroblastoma cells die more efficiently with combined antisense bcl-2/FLIP treatment in vitro than neuroblastoma cells with low MYCN protein levels (Poulaki, V., Proc. Am. Assoc. Cancer Res. 42:271, abstract, 2001). If this holds true in vivo, gene targeting therapies for bcl-2 and FLIP will be particularly beneficial in the very group of neuroblastoma patients who currently have the worst treatment failures.